The principal objectives of the proposed research are (a) to investigate the dynamics and structure of the DNA-dependent RNA polymerase, (b) to resolve the complex processes of RNA synthesis into their elementary steps, and (c) to elucidate the regulatory mechanism of gene transcription. We propose to approach these objectives by various physicochemical methods, in particular, the modern techniques of fast kinetics and nanosecond fluorescence spectroscopy. In this way, both structural and dynamic information will be yielded. Studies will be carried out primarily with the Escherichia coli system because large quantities of the highly purified RNA polymerase and the various components of the control system of lac operon can be obtained. The problems to be investigated include: (1) the structural and functional relationships among the multiple active sites of RNA polymerase, (2) the role of sigma subunit-core polymerase interaction in gene transcription, (3) the spatial organization and biological assembly of the subunits of RNA polymerase, (4) the kinetics and thermodynamics of the interactions between RNA polymerase, DNA template, and nucleoside triphosphates, and of the formation of phosphodiester bonds in RNA synthesis, (5) the syncatalytic mapping and the translocation of the enzyme during the course of RNA synthesis, and (6) the molecular mechanisms involved in the positive and negative control of transcription of the lac operon. The ultimate goal of this study is to understand the mechanism and regulation of gene expression in molecular detail. BIBLIOGRAPHIC REFERENCES: Conformational Transitions of the lac Repressor from E. coli, F. Y.-H. Wu, P. Bandyopadhyay, and C.W. Wu, J. Mol. Biol., 100, 459 (1976). The Interaction of a Subunit with Core Enzyme of E. coli RNA Polymerase, C.-W. Wu, F. Y.-H. Wu and L. R. Yarbrough, Biophys. J., 16, 15a (1976).